Fig. 6

Denervation-induced spine loss is mitigated by chronic light-activation of denervated GCs. (a) Semi-schematic diagram illustrating the entorhinal cortex lesion (ECL) in vitro model. The perforant pathway from the entorhinal cortex (EC) to the dentate gyrus (DG) is cut under optical control using a sterile blade and the EC is removed from the dish. In response to entorhinal denervation, GCs lose a significant portion of their dendritic spines. (b) Example of an OTC transduced with ChR2-tdTomato before ECL, immediately following ECL, and 4 days post lesion (dpl). (c) To test whether optogenetic activation of GCs can protect denervated GCs from spine loss, OTCs were subjected to four different conditions. One set of denervated and control cultures was not illuminated, whereas the other set of denervated and control cultures was chronically illuminated with 450 nm LEDs over four days using a low-frequency stimulation protocol (5 pulses with a pulse length of 10 ms at 12.5 Hz every hour). (d) A quantification of changes in spine density between day 0 (pre ECL) and 4 dpl showed a significant difference between non-illuminated control cultures and OTCs that have been denervated (two-way robust ANOVA with post hoc Brunner-Munzel tests, P = 0.002; Control: n = 16; ECL: n = 19), exhibiting the characteristic spine loss at 4 dpl. There was no significant difference between non-denervated control cultures that were not stimulated and those that were stimulated (n = 17) nor between denervated cultures that were not stimulated and those that were stimulated (n = 22). Importantly, there was also no significant difference between LED-stimulated OTCs that were non-denervated and those that had been denervated. These findings suggest that the effect of denervation was attenuated in cultures that were illuminated. Indeed, the effect of ECL depended on LED stimulation (ECL x stimulation interaction: WTS: 5.30; P = 0.024). Nevertheless, denervated OTCs that were stimulated were still significantly different from the non-illuminated, non-denervated control (post hoc Brunner-Munzel test, P = 0.037). n represents number of dendritic segments. Bars represent means ± SEM. *P < 0.05; **P < 0.01.